Hepatitis C virus (HCV) causes acute and chronic hepatitis and hepatocellular carcinoma (HCC). Notably, HCV has played a major role in the rise of HCC, accounting for 50% of cases in the United States. Despite an obvious need however, no vaccine is available to protect against HCV infection and only a subset of chronically infected patients respond to current treatment options. The major obstacle impeding HCV research has been a lack of cell culture and small animal infection models. While significant advancement has been made with the identification of a genotype 2a consensus clone that we and others have shown can produce infectious HCV in vitro in human hepatoma-derived Huh7 cells, a physiologically relevant, differentiated hepatocyte culture model is still needed to elucidate how HCV-induced alterations in hepatocyte function lead to HCV-associated liver disease, specifically the role the virus plays in malignant transformation of hepatocytes. As such, the objective of this proposal is to develop a human liver tissue model for the study of the molecular details involved in HCV infection and the development of HCV-associated HCC. Specifically, NASA-engineered bioreactors, termed rotating wall vessels (RWVs), will be utilized to establish a low-shear, low-turbulence environment that allows cells to co-localize spatially, grow three-dimensionally, and differentiate into highly specialized tissues. The hypotheses to be tested are that hepatocytes cultured in rotating wall vessels are functionally more similar to the liver in vivo and that these cells therefore will provide an improved system to study HCV pathogenesis, particularly HCC. Accordingly, the specific aims of this proposal are: 1) Characterize 3-D cultured Huh7 cells in the RWV to determine if these hepatoma cells can be induced to differentiate and down regulate their cancer-specific markers. 2) Establish primary human hepatocyte cultures three-dimensionally in the RWV and characterize whether they maintain a differentiated phenotype. 3) Evaluate and use three-dimensional Huh7 and primary human hepatocyte cultures for the study of HCV- host cell interactions and resulting cellular pathology, particularly the etiology of HCV-associated HCC. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) infects more than 170 million people worldwide, accounting for up to 50% of cases of hepatocellular carcinoma (HCC) in the United States. Because a physiologically relevant non-transformed, differentiated hepatocyte tissue culture model is needed to elucidate the role the virus plays in malignant transformation of hepatocytes, the overall objective of this proposal is to develop an advanced human liver tissue model for the study of the molecular details involved in HCV infection and the development of HCV-associated HCC.